Defects in the function of microvessels are responsible for much of the retinal injury that occurs in diabetes. Our long term goal is to determine the biochemical mechanisms by which retinal microvessels are damaged in diabetes. When exposed to high concentrations of glucose in tissue culture, retinal pericytes and endothelial cell ssynthesize sorbitol. We believe that intracellular sorbitol may interfere with receptor mediated turnover of inositol phospholipids and subsequent activation of protein kinase C and this may, in part, underlie the microangiopathy. To test this hypothesis we will expose retinal pericytes and endothelial cells in tissue culture to high concentrations of glucose and investigate their metabolism and transport of inositol, turnover of inositol phospholipids, actiatin of protein kinase C, and several aspects of cell function including sodium transport, prostaglandin release, and cell growth. Inositol entry into microvascular cells will be determined by studying the uptake of 3H-inositol. The intracellular concentration of inositol and sorbiotal will be determine as a function of external inositol and glucose concentrations. To measure alterations in the utrhover of inositol phospholipids, the cells will be labeled with 3H-inositol and the release of inositol phosphates as well as changes in the specific activity of inositol phospholipids in the cell membranes will be measured. The activation of protein kinase C by specific agonists will be determined in both the cytosol and membrane fractions of cells growing in normal and high concentrations of glucose. Mitogenesis will be assayed by the incorporation of thymidine into DNA. Sodium transport will be measured as amilioride sensitive Na-H exchange, ouabain sensitive K uptake, binding of ouabain to membrane fractions and sodium dependent uptake of amino acids. Formation of prostaglandins by the microvascular cells will be measured by radioimmunoassay. We will determine the effect of Sorbinil, an inhibitor of aldose reductase upon all functions altered by exposure of the cells to high concentrations of glucose.